Pharmaceutical composition and method for treatment or prevention of vascular disease or states of tissue hypoperfusion with hypoxic and/or ischemic consequences

ABSTRACT

Disclosed is the use of a substance selected from the group consisting of human apo-lactoferrin and/or peptides derivable from human lactoferrin and/or natural metabolites of human lactoferrin and/or functionally equivalent analogues of human apo-lactoferrin for the production of a pharmaceutical composition for treatment and/or prevention of a vascular disease and/or states of tissue hypoperfusion with hypoxic and/or ischemic consequences. Also a method for treatment of a vascular disease and/or states of tissue hypoperfusion with hypoxic or ischemic consequences wherein a therapeutically effective amount of a substance selected from the group consisting of human apo-lactoferrin and peptides derivable from human lactoferrin and natural metabolites of human lactoferrin and functionally equivalent analogues of human apo-lactoferrin is administered to a patient in need of said treatment is disclosed.

FIELD OF THE INVENTION

The present invention relates to the use of specific substances for theproduction of a pharmaceutical composition for treatment or preventionof vascular disease or states of tissue hypoperfusion with hypoxicand/or ischemic consequences. The invention also relates to a new methodfor treatment or prevention of vascular disease or states of tissuehypoperfusion with hypoxic and/or ischemic consequences.

BACKGROUND OF THE INVENTION

Lactoferrin (LF) is an 80 kD iron binding glycoprotein which mainly issynthesised by myelopoetic blood cells, such as neutrophil leucocytes,and secretory epithelial cells. LF has been identified in secretions,such as saliva, tears, bronchial secretions, urine, sperm, bile andpancreatic juice. Human milk contains approximately 1 g LF / litre andcolostrum (the first breast milk) approximately 7 g LF / litre. LF inbreast milk is mainly of apo type, i.e. iron-unsaturated or iron-freeLF. LF is also found in plasma but in considerably lower concentrationcompared to in secretion. LF is furthermore found in specific granulaein neutrophilic leukocytes, from where it is liberated as a response toinflammation.

LF has high affinity to receptors in various specific cell types,including the endothelial cells of the blood vessels. When iron binds toLF the crystallographic structure, and probably also the binding sites,of the molecule is changed, and thus also its function.

LF is considered to have many functions related to the defence of thehost, especially with regards to the immunological defence andantibacterial activity.

Bovine apo-LF (apo-bLF) and lactoferricin (bLFcin) have aftersubcutaneous and per oral administration, with somewhat differingresults, been shown to reduce the formation of both primary and daughtertumours in several experimental tumour systems in both mouse and rat.The mechanisms for this anti-tumour effect are not completely clear.

Angiogenesis, or as it is also called neovascularisation, is at thebeginning a purely microvascular reaction resulting in the formation ofnew microvessels from already existing microvessels (the smallestvenules and capillaries, respectively). The diameters of the newlyformed vessels may increase with time. Angiogenesis is a prerequisitefor the growth and metastatic spread of tumours.

Recently the inventor et al. reported for the first time that apo-bLFsignificantly suppresses VEGF₁₆₅ induced angiogenesis [Norrby K,Mattsby-Baltzer I, Innocenti M, Tuneberg S. (2001) Orally administeredbovine lactoferrin inhibits VEGF₁₆₅-mediated angiogenesis in the rat.Int J Cancer 91: 236-240].

Such a suppression of angiogenesis could be the reason behind theabove-mentioned inhibitory effects of bLF in tumour models.

VEGF₁₆₅ is a basic and heparin binding, 45 kD homodimeric glycoprotein.It is a unique endothelial specific mitogen and a pro-angiogenicmolecule produced by most cell types, in particular during hypoxia (lowoxygen pressure in the tissue) or ischemia (insufficient blood supplycausing hypoxia, further discussed below). High affinity receptors forVEGF₁₆₅ are almost exclusively located on endothelial cells, cells thatcover the walls of the blood vessels and have the ability to form newblood vessels.

The human gene for VEGF is found on chromosome 6 (6p21.3). Alternativeexon splicing of one single VEGF gene results in the formation of fourdifferent molecules with 121, 165, 189 and 206 amino acid residues,respectively. VEGF₆₅ is by far the most common isoform. Hypoxia, andglucose deficiency connected therewith, is the most efficacious factorfor upregulation of VEGF. Hypoxia-inducible factor, HIF, is a keyregulator of responses to hypoxia inducing the VEGF gene. Also thenumber of specific VEGF receptors on the endothelial cells is increasedduring hypoxia.

During insufficient local blood supply, ischemia, both hypoxia andinflammation occurs. Impaired blood circulation, ischemia, can causeserious clinical symptoms in e.g. the cardiac muscle resulting in anginapectoris and/or myocardial infarction, in the brain resulting in strokeor cerebral infarction, or in the lower limbs resulting ultimately ingangrene.

Significant physiologic angiogenesis occurs hardly ever in adults, withthe exception for in the ovaries, the endometrium, and the placenta.Angiogenesis is however a rapid, life sustaining reaction in woundhealing and inflammation. There is thus an exceptionally powerfulregulation of the balance between pro- and anti-angiogenic factors inthe body. The balance is upheld by an apparently large, not yet fullyelucidated, system of pro- and anti-angiogenic endogenous molecules.

SUMMARY OF THE INVENTION

As stated above, it was recently reported that bovine apo-LFsignificantly inhibits VEGF₁₆₅ induced angiogenesis. Very unexpectedlyand surprisingly, the inventor has now found, in exactly the sameexperimental system, that human apo-LF on the contrary stimulatesVEGF₁₆₅ induced angiogenesis. Human apo-LF has thus a pro-angiogeniceffect, which can be used for therapeutic angiogenesis.

Thus, the present invention relates to the systemic or local use ofhuman apo-lactoferrin and/or peptides derivable from humanapo-lactoferrin, natural metabolites thereof and/or functionallyequivalent analogues thereof in order to enhance VEGF inducedangiogenesis to prevent tissue damage. In vascular disease such tissuedamage is typically caused by occlusive or non-occlusive vasculardisease leading to tissue hypoxia or ischemia.

The clinical effects of such tissue hypoxia or ischemia may beexemplified by impending or manifested infarction such as myocardialinfarction, stroke or gangrene. Another important vascular diseaserelated to such hypoxic/ischemic states is angina pectoris. Otherclinical conditions that would benefit from enhanced VEGF inducedangiogenesis are various types of wound healing situations such as inpeptic ulcers and leg ulcers. Certain types of male hair loss, such asandrogenic alopecia, might also be a condition where enhancedangiogenesis might be beneficial.

A common denominator for all these ischemic and/or hypoxic conditions isthe resulting local expression or biological effect of VEGF. Accordingto the present invention the use of human apo-lactoferrin and/orpeptides derivable from human apo-lactoferrin, natural metabolitesthereof and/or functionally equivalent analogues thereof, enhancesangiogenesis. An attractive, but not binding, explanation for this isthat these substances enhance and/or stimulate the expression or thebiological effects of VEGF.

One objective of the present invention is thus the use of a substanceselected from the group consisting of human apo-lactoferrin and/orpeptides derivable from human lactoferrin and/or natural metabolites ofhuman lactoferrin and/or functionally equivalent analogues of humanapo-lactoferrin for the production of a pharmaceutical composition fortreatment and/or prevention of a vascular disease or states of tissuehypoperfusion with hypoxic and/or ischemic consequences.

Another object of the present invention is a method for treatment orprevention of a vascular disease or states of tissue hypoperfusion withhypoxic and/or ischemic consequences where in a therapeuticallyeffective amount of a substance selected from the group consisting ofhuman apo-lactoferrin and/or peptides derivable from human lactoferrinand/or natural metabolites of human lactoferrin and/or functionallyequivalent analogues of human apo-lactoferrin is administered to apatient in need of said treatment.

The characterizing features of the invention will be evident from thefollowing description and the appended claims.

DETAILED DESCRIPTION OF THE INVENTION

As stated above, the invention is based on the use of a substanceselected from the group consisting of human apo-lactoferrin and/orpeptides derivable from human apo-lactoferrin and/or natural metabolitesof human apo-lactoferrin and/or functionally equivalent analogues ofhuman apo-lactoferrin.

The lactoferrin used according to the invention may be of any isoform.It may also be recombinant human apo-lactoferrin.

Said natural metabolites may for example be lactoferricin, i.e. apepsin-cleaved fragment from human lactoferrin or hydrolysates of humanlactoferrin.

Said functionally equivalent analogues are substances that arestructurally similar to human apo-lactoferrin or to natural metabolitesthereof, which have essentially the same pro-angiogenic effect as humanapo-lactoferrin.

Said peptides derivable from human lactoferrin may be fragments ormodified fragments obtainable from human lactoferrin. They may be eithernaturally occurring or synthetically produced peptides. They may forexample be the peptides described in the International application withpublication number WO 00/01730, which is incorporated herein byreference. In particular, the peptides disclosed in the sequence listingof WO 00/01730 are suitable for use according to the present invention.These include peptides constituted of all or some of the amino acids12-40 of human lactoferrin counted from the N-terminal end, andpreferably modified versions thereof. More specifically they include thepeptides formed of the sequences constituted of amino acids 16-40 andamino acids 18-40 from the N-terminal end of human lactoferrin, withsome alterations, and also sequences with only 14 residues, roughlycorresponding to residues 18-31 of human lactoferrin wherein C-20 isreplaced by A, Q-22 is replaced by K, and N-26 is replaced by D. Theyalso include peptides formed of the amino acids in positions 12-31,counted from the N-terminal end, in the sequence constituting humanlactoferrin, as well as modifications thereof, and also fragments ofthis sequence consisting of at least 7 amino acids. Furthermore, theyinclude peptides consisting of 11-17 amino acids corresponding to thesequences that begin with one of the amino acids in positions 15-21 andend with the amino acid in position 31, counted from the N-terminal end,in the sequence constituting human lactoferrin, as well as modificationsthereof. Moreover, they include peptides consisting of 12 amino acidsbased on the sequence consisting of the amino acids in positions 20-31in human lactoferrin, counted from the N-terminal end.

The substances and pharmaceutical compositions can according to theinvention be used for all medical disorders that benefit fromstimulation of VEGF induced angiogenesis, or diseases where there is aninsufficient effect of VEGF either caused by increased effects ofanti-angiogenic factors, such as certain hormones, or caused byinsufficient production of VEGF. By insufficient effect of VEGF orinsufficient production of VEGF is intended that angiogenesis does notoccur sufficiently for prevention or improvement of a disease.

For the purpose of this disclosure, the terms “illness”, “disease”,“medical condition”, “abnormal condition” and the like can be usedinterchangeably with “medical disorder”. Examples of disorders thatbenefit from the treatment according to the invention, i.e. theenhancement of VEGF stimulated angiogenesis, are conditions of hypoxiaand/or ischemia resulting in angina pectoris, impending or manifestedmyocardial infarction, stroke or gangrene, wounds, such as peptic or legulcers that are slow in healing, and certain types of hair loss.

The term “treatment”, as it is used herein, relates to both treatment inorder to cure or alleviate a disease or a condition, and to treatment inorder to prevent the development of a disease or a condition. Thetreatment may either be performed in an acute or in a chronic way.

VEGF is upregulated virtually exclusively under conditions of hypoxiaand is the key angiogenic factor linking ischemia and collateralcompensatory angiogenesis. In hypoxia the degradation of thetranscription factor hypoxia-inducible factor (HIF-1 alpha) issuppressed leading to activation of the hypoxia responsive elements andsubsequently the VEGF gene. As stated above, the substances andpharmaceutical compositions according to the invention are suitable fortreatment of tissue ischemia. VEGF₁₆₅ is over expressed during tissueischemia, i.e. locally decreased oxygen pressure due to insufficientoxygen supply due in turn to impaired blood circulation. During ischemiathe effect of VEGF₁₆₅ is considered to be necessary for localcompensatory collateral angiogenesis, which leads to locally increasedblood supply. If ischemic conditions are not reversed they can result ininfarction, i.e. tissue death. This can happen in e.g. the cardiacmuscle (myocardial infarction), the brain (brain infarction or stroke)or in distant parts of the lower limbs, such as toes, the feet or thelower part the leg (gangrene) resulting in severe clinical consequences.Stimulation of the VEGF₁₆₅ mediated angiogenesis with the substancesaccording to the invention reduces the risk of i.a. myocardialinfarction, stroke and gangrene.

The substances and pharmaceutical compositions according to theinvention are thus suitable for treatment of cardiovascular disease,especially coronary and carotid artery disease. Cardiovascular diseasecaused by vascular damage, endothelial dysfunction and atheroscleroticchanges is the most common cause of death in the western world.Atherosclerosis is in particular common in connection with diabetesmellitus. The occurrence of diabetes, and in particular diabetes typeII, increases dramatically globally, probably due to changed ways oflife.

Furthermore, the substances and pharmaceutical compositions according tothe invention are also suitable for treatment of peripheral arteryocclusive disease, PAOD, which is caused by atherosclerosis and affectsprimarily the lower limbs. Advanced cases of PAOD cannot be treated byreconstructive-, angioplastic- or by pass-surgery. The use of thesubstances according to the invention may prevent the need of amputationor angiogenic gene therapy in impending gangrene. Current clinicaltrials with therapeutic angiogenesis is essentially based on theadministration of the gene of an angiogenic factor, such as VEGF₁₆₅,with the aid of viral or plasmid vector systems. These techniques havelimitations and complications, e.g. it is difficult to achieve thecorrect dosage and not only endothelial cells within the hypoxic tissueare affected [Ferber, D. (2001) Gene therapy: Safer and virus-free?Science 295:1638-42]. Several gene therapy clinical trials are currentlygoing on worldwide, but so far no such treatment has been approved forclinical use.

The use of the substances or pharmaceutical compositions according tothe invention thus provides a new way to treat PAOD. According to theinvention it is possible to replace the use of currently tested types oftherapeutic angiogenesis with the use of the substances orpharmaceutical compositions according to the invention. One advantage isthat this treatment may be given continuously, or repeatedly, duringlong periods of time without or with limited side effects, since thistreatment is targeted in that only ongoing VEGF mediated angiogenesis isstimulated.

The pharmaceutical composition, or medicinal product, according to theinvention may also comprise other substances, such as an inert vehicle,or pharmaceutically acceptable adjuvant, carriers, preservatives etc,which are well known to persons skilled in the art.

In the method according to the present invention a therapeuticallyeffective amount of the above substance is administered to the patient.The expression “therapeutically effective amount” relates to an amountthat will lead to the desired therapeutic effect, i.e. an amount thatwill enhance the VEGF mediated angiogenesis.

The substance or pharmaceutical composition according to the inventionis administered to the patient orally, parenterally, locally and/or byinhalation. For local administration the pharmaceutical composition orthe substance may e.g. be in the form of an ointment, a solution or aspray. The term “patient”, as it is used herein, relates to any human ornon-human mammal in need of treatment according to the invention, i.e.any human or non-human mammal that benefits from VEGF inducedangiogenesis or therapeutic angiogenesis.

The invention will now be further explained in the following examples.These examples are only intended to illustrate the invention and shouldin no way be considered to limit the scope of the invention.

EXAMPLES

Methods

The method used herein to study the formation of new microvessels, “therat mesenteric-window angiogenesis assay”, has the following advantagescompared to other models of angiogenesis: (1) Similar to almost alltissues in human adults the test tissue used is natively vascularisedand lacks physiological angiogenesis. (2) No surgical procedures, whichinevitably cause a wound healing reaction accompanied by angiogenesis,are used. (3) The method allows for a good quantification of theangiogenic response in objective parameters.

In order to induce angiogenesis the animals are given repeated picomolardoses of recombinant VEGF₁₆₅ intra peritoneally, which quickly reachesthe test tissue.

When the animal has been sacrificed, the intact test tissue is studiedas spread preparations on objective slides, which is a major advantagecompared to the study of the presence of vessels in microtome-sectionedtissue. Several of the parameters are measured by interactive imageanalysis techniques. A number of relevant, objective parameters aremeasured using light microscopic morphometry with high accuracy. Byusing purified anti-rat endothelium monoclonal antibody CL 043AP alsothe smallest microvessels are visualized immunocytochemically [Norrby K,Mattsby-Baltzer I, Innocenti M, Tuneberg S. (2001) Orally administeredbovine lactoferrin inhibits VEGF₁₆₅-mediated angiogenesis in the rat.Int J Cancer 91: 236-240]. By using this method, it is possible toadminister modulators of angiogenesis systemically, which simulates aclinical therapeutic situation.

A measurement of spatial distribution of the vessel network is“vascularised area” (VA) expressed in % of the whole area permesenterial window [Norrby K. (1994) Basic fibroblast growth factor andmammalian de novo angiogenesis. Microvasc Res 48: 96-113; Norrby K.(1996) Vascular endothelial growth factor and mammalian de novoangiogenesis. Microvasc Res 51: 153-163].

Microvascular density is measured as “microvascular length”, MVL(percentage pixles occupied by vessels made 1 pixle thick); “totalmicrovascular length” (TMVL)=VA×MVL [Norrby K. (1994) Basic fibroblastgrowth factor and mammalian de novo angiogenesis. Microvasc Res 48:96-113; Norrby K. (1996) Vascular endothelial growth factor andmammalian de novo angiogenesis. Microvasc Res 51: 153-163].Microvascular density and pattern formation is measured in terms of thelength of the individual microvascular segments (Le. MS, length ofmicrovessel segment, the distance between two subsequent branchingpoints) and the number of them (No. MS, number of microvessel segments)per volume unit of tissue, as well as the microvessel tortuosity (MVT)[Norrby K. (1998) Microvascular density in terms of number and length ofmicrovessel segments per unit tissue volume in mammalian angiogenesis.Microvasc Res 55:43-53]. The method for measurement of the frequency ofmicrovascular loop formation (In. LF, index of loop formation),intersection (In. IS, index of intersection) and the number of branchingpoints (No. BP) per volume unit of tissue was also used [Näslund I,Norrby K. (2000) NO and de novo mammalian angiogenesis: further evidencethat NO inhibits bFGF-induced angiogenesis while not influencingVEGF₁₆₅-induced angiogenesis. APMIS 108: 29-37; Norrby K. (2000) 2.5 kDand 5.0 kD heparin fragments specifically inhibit microvessel sproutingand network formation in VEGF₁₆₅-mediated mammalian angiogenesis. Int JExp Path 81: 191-198]. Le. MS is influenced by several factors, such asthe ability of the microvascular segments to increase their length, todivide and to form interconnecting loops.

The number of microvessel sprouts per unit tissue volume (No. SP), theirlength (Le. SP) and degree of tortuosity (SPT) were measured at the edgeof the network in one of the experiments (Table 4), as describedelsewhere [Näslund I, Norrby K. (2000) NO and de novo mammalianangiogenesis: further evidence that NO inhibits bFGF-inducedangiogenesis while not influencing VEGF₁₆₅-induced angiogenesis. APMIS108: 29-37; Norrby K. (2000) 2.5 kD and 5.0 kD heparin fragmentsspecifically inhibit microvessel sprouting and network formation inVEGF₁₆₅-mediated mammalian angiogenesis. Int J Exp Path 81: 191-198].Increase in No. SP indicates increased microvessel proliferation. TheLe. SPs were ranked in order of size in each treatment group.

Example 1

In this experiment the effect of ingested apo-hLF on VEGF₁₆₅ mediatedangiogenesis was demonstrated. The results are shown below in Table 1.

Lactoferrin, dissolved in saline, was given by tube feeding twice dailyfrom Sunday afternoon (Day -1) to Friday afternoon (Day4). Vehiclecontrols received saline by tube feeding. The angiogenic treatment withVEGF was given i.p. on Days 0-4 (twice daily). TABLE 1 Effect of orallyadministered iron-unsaturated human lactoferrin (20 mg/kg twice daily),apo-hLF, on VEGF₁₆₅ mediated angiogenesis VEGF treated + VEGF treated +% of Untreated vehicle apo-hLF vehi- controls Variable n = 14 n = 14 clen = 8 Microvessel proliferation VA  7.82 ± 1.93 12.09 ± 1.49^(a) 1551.18 ± 0.50 MVL 1.085 ± 0.137 1.465 ± 0.077^(a) 135 0.28 ± 0.04 TMVL 8.49 ± 2.10 17.72 ± 2.19^(b) 209 0.33 ± 0.14 No. BP 238.9 ± 39.7 376.7± 31.7^(b) 158 32.5 ± 6.7  No. MS 279.0 ± 49.4 457.6 ± 40.8^(b) 164 32.7± 7.4  Le. MS  7-17  5-15^(d) \  8-19 (0-10) (range, μm) Le. MS 178-534157-668^(d) \ 265-719 (90-100) (range, μm) Le. MS 61.5 55.1  90 78.1median, μm Microvessel network pattern formation MVT  7.39 ± 0.34 (8) 7.54 ± 0.14 (12) 102 4.70 ± 0.73 In. LF 1.144 ± 0.014 1.208 ± 0.008^(c)106 0.988 ± 0.031 In IS 13.89 ± 1.23 15.68 ± 0.87 112 10.20 ± 2.58 ^(a)p ≦ 0.05;^(b)p ≦ 0.01;^(c)p ≦ 0.002;^(d)p ≦ 0.0001 compared with vehicle controls.\ indicates statistically significant shortening of the Le. MS.Figures in parentheses indicate number of animals, if less than 14 (orif less than 8 in the case of untreated controls), analysed according tothe set criteria.The distribution of Le. MS, the (0-10) percentile, the (90-100)percentile, and the median value, was based on thousands of individuallymeasured microvessel segments in both vehicle controls and in the groupof animals treated with apo-hLF.Mean ± SEM.

VA, MVL, TMVL, No. BP, No. MS and MVT increased statisticallysignificantly in the animals receiving VEGF i.p. and saline orally ascompared to the untreated controls, thus demonstrating the proangiogeniceffect of the VEGF treatment.

Treatment with apo-LF further increased these measurements, indicatingan additional stimulatory effect of apo-hLF on VEGF-stimulatedangiogenesis.

The results demonstrate that oral administration of apo-hLFsignificantly enhanced the VEGF mediated angiogenic response.

Comparative Example 1

In this comparative experiment it was demonstrated that ingestedholo-hLF, in contrast to apo-hLF, does not have any significant effecton VEGF₁₆₅ mediated angiogenesis. The results are shown below in Table2.

Groups and treatments were as described under Example 1, Table 1, apartfrom that apo-hLF was replaced by holo-hLF. TABLE 2 Effect of orallyadministered iron-saturated human lactoferrin (20 mg/kg twice daily),holo-hLF, on VEGF₁₆₅ mediated angiogenesis. VEGF-treated +VEGF-treated + % of Untreated vehicle holo-hLF vehi- controls Variable n= 14 n = 14 cle n = 8 Microvessel proliferation VA 5.95 ± 0.73  5.77 ±1.12 97 1.38 ± 0.38 MVL 1.110 ± 0.079 1.067 ± 0.121 96 0.381 ± 0.053TMVL 6.80 ± 0.98  6.45 ± 1.20 95 0.53 ± 0.14 No. BP 242.6 ± 20.4  241.1± 31.7 99 62.4 ± 12.4 No. MS 286.0 ± 25.1  289.0 ± 39.7 101 68.7 ± 15.6Le. MS  5-17  5-15^(a) \  5-16 (0-10) (range, μm) Le. MS 174-710 172-576221-458 (90-100) (range, μm) Le. MS 60.2 58.9 98 median, μm Microvesselnetwork pattern formation MVT 8.36 ± 0.29  9.13 ± 0.34 (12) 109 6.95 ±0.39 (13) (3) In LF 1.174 ± 0.007 1.183 ± 0.015 101 1.054 ± 0.051 In IS11.71 ± 1.04  13.00 ± 0.96 111 14.24 ± 1.70 p ≦ 0.05 compared with vehicle controls of the same experiment\ indicates statistically significant shortening of the Le. MS comparedwith vehicle controlFigures in parentheses indicate number of animals, if less than 14 (orif less than 8 in the case of untreated controls), analysed according tothe set criteria.The distribution of Le. MS, the (0-10) percentile, the (90-100)percentile, and the median value, was based on several thousandsindividually measured microvessel segments in both vehicle controls andin the group of animals treated with holo-hLF.Mean ± SEM.

VA, MVL, TMVL, No. BP, No. MS and MVT increased statisticallysignificantly in the animals receiving VEGF i.p. and saline orally ascompared to the untreated controls, thus demonstrating the proangiogeniceffect of the VEGF treatment.

Unlike with apo-hLF, treatment with holo-hLF did not increase theVEGF-stimulated angiogenesis.

Comparative Example 2

Besides VEGF, bFGF (basic fibroblast growth factor) is probably the mostimportant angiogenic factor. It was thus of interest to test whetherapo-hLF also affected bFGF stimulated angiogenesis.

In this comparative experiment it was demonstrated that apo-hLF does nothave any significant effect on bFGF-mediated angiogenesis, in contrastto its effect on VEGF₁₆₅ mediated angiogenesis. The results are shownbelow in Table 3.

The same experimental conditions were used as in the experiments withVEGF induced angiogenesis. TABLE 3 Effect of orally administerediron-unsaturated human lactoferrin (20 mg/kg twice daily), apo-hLF, onbFGF mediated angiogenesis bFGF-treated + BFGF-treated + Untreatedsaline holo-hLF % of controls Variable n = 14 n = 14 vehicle n = 8Microvessel proliferation VA 7.79 ± 0.98  7.21 ± 0.88 93 4.49 ± 1.01 MVL1.049 ± 0.090 1.226 ± 0.068 117 0.514 ± 0.053 TMVL 8.17 ± 1.03  8.84 ±1.08 108 2.30 ± 0.52 No. BP 252.9 ± 32.4  274.8 ± 18.7 109 86.7 ± 12.7No. MS 302.5 ± 42.1  322.6 ± 23.4 107 95.0 ± 15.7 Le. MS  5-12  5-13 — 5-18 (0-10) (range, μm) Le. MS 159-530 172-716^(a) \ 192-485 (90-100)(range, μm) Le. MS 55.9 60.8 109 70.6 median, μm Microvessel networkpattern formation MVT 8.90 ± 0.17  9.35 ± 0.20 105 8.66 ± 0.43 (13) (5)In. LF 1.178 ± 0.014 1.168 ± 0.010 99 1.075 ± 0.027 In IS 15.13 ± 0.91 13.52 ± 1.18 89 9.16 ± 1.55^(a)p ≦ 0.0001 compared with vehicle control./ indicates statistically significant lengthening of the Le. MS.—indicates statistically insignificant change of Le. MS.Figures in parentheses indicate number of animals, if less than 14 (orif less than 8 in the case of untreated controls), analysed according tothe set criteria.The distribution of Le. MS, the (0-10) percentile, the (90-100)percentile, and the median value, was based on thousands of individuallymeasured microvessel segments in both vehicle controls and in the groupof animals treated with apo-hLF.Mean ± SEM.

VA, MVL, TMVL, No. BP and No. MS increased significantly in statisticalterms in the animals receiving bFGF i.p. and saline orally as comparedto the untreated controls, thus demonstrating the pro-angiogenic effectof the bFGF treatment.

It was found (see Table 3) that bFGF mediated angiogenesis is notaffected by apo-hLF, apart from the fact that the longer microvascularsegments (Le. MS, see above) were significantly lengthened. The meaningof this is not clear, and one should note that the correspondingpopulation or Le. MS were significantly shortened by apo-hLF in VEGFmediated angiogenesis (see Table 1).

These results indicate that enhancing effect of apo-hLF seen in VEGFmediated angiogenesis is specific and not valid for angiogenesis ingeneral, which is very interesting both theoretically and clinically.

Comparative Example 3

In this comparative experiment it was demonstrated that continuouslys.c. infused apo-hLF signficantly enhanced VEGF165-mediated angiogenesis(Table 4) and the number of capillary sprouts (Table 5). TABLE 4 Effectof subcutaneously continuously infused iron-unsaturated humanlactoferrin, apo-hFL, on VEGF164-mediated angiogenesis. Vascularizedarea (VA), microvascular length (MVL) and total microvascular length(TMVL) Apo-hLF Vehicle 2 mg/kg/24 hr 20 mg/kg/24 hrs Variable n = 14 n =11 n = 12 VA 8.04 ± 1.53 12.35 ± 2.46  9.71 ± 1.60 (100%) (154%) (121%)MVL 1.004 ± 0.100 1.260 ± 0.120a 1.269 ± 0.113a (100%) (125%) (126%)TMVL 8.04 ± 1.54 15.55 ± 3.09b 12.32 ± 2.03 (100%) (193%) 153%)Lactoferrin, dissolved in saline, was infused s.c. for 7 days, startingon Day - 1, using an Alzet ® osmotic pump. Vehicle controls receivedsaline s.c. by the same type of pump. The angiogenic treatment with VEGFwas given i.p. on Days 0-4. The animals were sacrificed on Day 8.ap ≦ 0.05;bp ≦ 0.05 compared with vehicle controls. In addition, the differencebetween control and the combined data of the low and the high dose ofapo-hLF was significant in terms of MVL and TMVL (p ≦ 0.05).Number of animals, n.Mean ± SEM.

TABLE 5 Effect of subcutaneously continuously infused humaniron-unsaturated lactoferrin, apo-hLF, on VEGF₁₆₄-mediated angiogenesis.Number of microvessel sprouts per unit tissue volume (No. SP), sproutlength (Le. SP) and degree of sprout toruosity (SPT) Apo-hLF Vehicle 2mg/kg/24 hr Variable n = 14 n = 11 No. SP 107.1 ± 9.3 133.4 ± 9.2^(a)(100%) (125%) Le. SP, 0-10 percentile 8-72  14-66^(b) (−) (range, μm)Le. SP, 90-100 percentile 482-1237 441-1042^(c) (−) (range, μm) Le. SP,median, μm 200.1 168.9 (100%) (84%) SPT (A.U.)  10.0 ± 0.3  11.0 ±0.3^(a) (100%) (110%)

Lactoferrrin, dissolved in saline, was infused for 7 days, starting onDay -1, using an Alzet® osmotic pump. Vehicle controls received salines.c. by the same type of osmotic pump. The angiogenic treatment withVEGF was given i.p. on Days 0-4. The animals were sacrificed on Day 8.The variables were measured at the edge of the microvascular network inthose mesenteric windows that were also analyzed in regard to a numberof other angiogenesis variables within the same network, as presented inTable 3.

The distribution of Le. SP (the 0-10 percentile, the 90-100 percentileand the median) was based on approximately 1,500 individually measuredsprouts in vehicle controls. Statistically significant shortening of theLe. MS compared with vehicle control, (−).

a) p ≦0.05, b) p ≦0.01, and c) p ≦0.0001 compared with vehicle controls.

Conclusions from the Examples

To summarize, the results presented in the examples and in Tables 1-5show that the oral or s.c. administration of apo-hLF specificallyenhances VEGF₁₆₅ mediated angiogenesis in the rat mesenteric-windowangiogenesis assay.

This effect seems to be specific for VEGF mediated angiogenesis sincebFGF mediated angiogenesis was not affected by apo-hLF. Also, thestimulatory effect of LF on VEGF mediated angiogenesis seems to bespecific for the apo-form since oral administration of holo-hLF did notaffect the measured angiogenic parameters.

1-32. (canceled)
 33. A method for treatment or prevention of a vasculardisease or states of tissue hypoperfusion with hypoxic or ischemicconsequences in a patient, the method comprising the following steps:selecting a substance from the group consisting of humanapo-lactoferrin, human lactoferricin, peptides derivable from humanlactoferrin, natural metabolites of human lactoferrin, and functionallyequivalent analogues of human apo-lactoferrin; determining atherapeutically effective amount of the selected substance;administering the therapeutically effective amount of the selectedsubstance to the patient whereby the method is used as an alternative tobypass surgery or any therapeutic angiogenesis options.
 34. The methodas recited in claim 33 whereby the method is used for treating impendingstroke, manifested stroke, ischemic heart disease such as anginapectoris or impending or manifested myocardial infarction, andperipheral artery occlusive disease with or without impending gangrene.35. The method as recited in claim 33 whereby the method is used fortreating vascular disease, state of tissue hypoperfusion, or state ofdepressed VEGF induced angiogenesis associated with peptic ulcer, legulcer or local or generalised hair loss.
 36. The method as recited inclaim 33 wherein said substance is administered orally.
 37. The methodas recited in claim 33 wherein said substance is administeredparenterally.
 38. The method as recited in claim 33 wherein saidsubstance is administered locally.
 39. The method as recited in claim 33wherein said substance is administered by inhalation.
 40. A method fortreatment or prevention of a vascular disease or states of tissuehypoperfusion with hypoxic or ischemic consequences in a patient, themethod comprising the following steps: selecting a peptide from thegroup consisting of peptides derivable from human lactoferrin;determining a therapeutically effective amount of the selectedsubstance; administering the therapeutically effective amount of theselected substance to the patient whereby the method is used as analternative to bypass surgery or any therapeutic angiogenesis options.41. The method as recited in claim 40 wherein the peptide comprises apeptide constituted of all or some of the amino acids 12-40 of humanlactoferrin counted from the N-terminal end, or a modified versionthereof.
 42. The method as recited in claim 40 wherein the peptidecomprises a peptide formed of the sequences constituted of amino acids16-40 and amino acids 18-40 from the N-terminal end of humanlactoferrin, or a modified version thereof.
 43. The method as recited inclaim 40 wherein the peptide comprises a peptide essentiallycorresponding to residues 18-31 of human lactoferrin wherein C-20 isreplaced by A, Q-22 is replaced by K, and N-26 is replaced by D.
 44. Themethod as recited in claim 40 wherein the peptide comprises a peptideformed of the amino acids in positions 12-31, counted from theN-terminal end, in the sequence constituting human lactoferrin, or amodification thereof, or a fragment thereof consisting of at least 7amino acids.
 45. The method as recited in claim 40 wherein the peptideis a peptide consisting of 11-17 amino acids corresponding to thesequences that begin with one of the amino acids in positions 15-21 andend with the amino acid in position 31, counted from the N-terminal end,in the sequence constituting human lactoferrin, or a modificationthereof.
 46. The method as recited in claim 40 wherein the peptide is apeptide consisting of 12 amino acids based on the sequence consisting ofthe amino acids in positions 20-31 in human lactoferrin, counted fromthe N-terminal end.
 47. The method as recited in claim 40 whereby themethod is used for treating impending stroke, manifested stroke,ischemic heart disease such as angina pectoris or impending ormanifested myocardial infarction, and peripheral artery occlusivedisease with or without impending gangrene.
 48. The method as recited inclaim 40 whereby the method is used for treating vascular disease, stateof tissue hypoperfusion, or state of depressed VEGF induced angiogenesisassociated with peptic ulcer, leg ulcer or local or generalised hairloss.
 49. The method as recited in claim 40 wherein said substance isadministered orally.
 50. The method as recited in claim 40 wherein saidsubstance is administered parenterally.
 51. The method as recited inclaim 40 wherein said substance is administered locally.
 52. The methodas recited in claim 40 wherein said substance is administered byinhalation.